Abstract - Fractionating Organelle Subpopulations by Size and Type Intracellular organelle heterogeneity in size and function is intimately associated with multiple dynamic processes, including fusion, fission, biomolecular synthesis and storage within the organelle, biomolecular transport, oxidative stress, and degradation (e.g. via mitophagy). Disease, aging, and drug treatment, perturbing the homeostatic balance of such processes, affect each organelle type differently and for a given type may result in organelle subpopulations with distinct size, function, or morphology. It is thus imperative to isolate organelles of specific type and, when present, their subpopulations, because this is the first step in characterizing their molecular composition, which is essential to decode alterations in function and molecular pathways that are obscured by uncertain subcellular localization. However, the most common techniques are not capable to isolate organelles based on size. Moreover, high purity organelle isolations require multiple, cumbersome and time consuming processes prone to sample loss and still suffer from organelle co-isolation exhibiting similar physical properties. Important biomolecular studies that must account for subcellular localization and that rely on sample quality, are thus severely limited by the lack of suitable technologies for size-based organelle subpopulations and functionally distinct organelles. This study will close this bottleneck by developing a novel fractionation technology capable of separating organelles by size and type in sufficiently large amounts and with high purity. The novel, cutting-edge microfluidic technology to fractionate organelles is based on migration mechanisms that occur under non-equilibrium conditions, require tailored microenvironments and tailored driving forces. If correctly designed these ?ratchet? devices exhibit unique selectivity for organelles by size and type, speed, and high throughput capabilities, here realized through a subtle interplay of electrokinetic and dielectrophoretic forces as well as the microfluidic device geometry. Numerical modeling tools will be developed based on experimentally observed migration parameters in specific aim (SA) 1. This in silico study is necessary since ratchet devices often follow ?non-intuitive? migration schemes and require detailed parameter studies to adapt them for biological applications. The optimized parameter set obtained from numerical modeling will then be experimentally validated for wild type (normal) and small as well as enlarged mitochondria generated via gene knock-out as a model for size-based separation in SA2. Wild type mitochondria as well as acidic organelles will serve as the model system for type-based separation (SA2). The novel technology will then be scaled-up to build a device for high throughput fractionation and collection of organelle fractions of different sizes or types, allowing the investigation of the phenotype of fractionated mitochondria subpopulations and highly pure organelle isolations with standard characterization methods (SA3). With the successful development of the novel fractionation technology, this project will provide a unique and pivotal tool for future inquiry into highly pure organelle fractions to unravel biological disease pathways in which organelle size and type play a critical role.